Dispersion and Retention of Dusts Consisting of Ultraﬁne Primary Particles in Lungs

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Consisting of Ultraﬁne Primary Particles in Lungs

D. Schaudien, J. W. Knebel, I. Mangelsdorf, J.-U. Voss, W. Koch, O. Creutzenberg

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Research Project F 2133

D. Schaudien J. W. Knebel I. Mangelsdorf J.-U. Voss W. Koch O. Creutzenberg

Dispersion and Retention of Dusts Consisting of Ultrafine Primary Particles in Lungs

Dortmund/Berlin/Dresden 2011

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This publication is the final report of the project “Dispersion and Retention of Dusts Consisting of Ultrafine Primary Particles in Lungs” – Project F 2133 – on behalf of the Federal Institute for Occupational Safety and Health.

The responsibility for the contents of this publication lies with the authors.

Authors: Dirk Schaudien, PhD Dr. Jan Wolfgang Knebel Dr. Inge Mangelsdorf Dr. Jens-Uwe Voss Prof. Dr. Wolfgang Koch Dr. Otto Creutzenberg

Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM)

Nikolai Fuchs Str. 1, 30625 Hannover, Germany Telephone +49 511 5350-461

Fax +49 511 5350-155 Project Manager: Dr. Otto Creutzenberg

Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM)

Cover photo: Sabine Plitzko

Federal Institute for Occupational Safety and Health Cover design: Rainer Klemm

Federal Institute for Occupational Safety and Health Publisher: Federal Institute for Occupational Safety and Health

Friedrich-Henkel-Weg 1-25, 44149 Dortmund, Germany Telephone +49 231 9071-0

Fax +49 231 9071-2454 poststelle@baua.bund.de www.baua.de

Berlin:

Nöldnerstr. 40-42, 10317 Berlin, Germany Telephone +49 30 51548-0

Fax +49 30 51548-4170 Dresden:

Fabricestr. 8, 01099 Dresden, Germany Telephone +49 351 5639-50

Fax +49 351 5639-5210

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Dispersion and Retention of Dusts Consisting of Ultrafine Primary Particles in Lungs

Abstract

This project aimed at studying the dispersion and retention behavior of dusts consisting of nanoscaled primary particles. Toxicological studies have demonstrated that the effects observed for nanoscaled particles are better correlated to the particle surface or particle number than to the administered mass doses. The toxicokinetic fate of nanoscaled particles and the potential effects induced after deposition in lungs are predominantly determined by the agglomeration status. Sytemic particle effects, i.e.

effects on the remote organs, in addition to those on the target organ respiratory tract are conceivable only for particles with a nanoscaled aspect.

In this study various types of nanoscaled particles, i.e titanium dioxide, carbon black, constantan and zinc oxide were dispersed in physiologically compatible media or generated as aerosols with well-defined characteristics. For aqueous nanoparticle suspensions, the hydrodynamic mean diameter and the ζ potential were determined, for aerosols the particle number or mass concentrations and the mass median aerodynamic diameter (MMAD). For aqueous formulation of nanoparticles, phosphate buffer, sometimes including auxiliaries such as bovine serum albumin (BSA) or Tween^® 80 (non-ionic surfactant), was used.

In an in vitro approach selected human bronchial and alveolar epithelial cell lines as well as fibroblasts grown on membranes were exposed from the apical side to the different particle types. After 1 hour the particles were detected by TEM technique in particular on the cellular surface whereas after 24 hours they were predominantly located in the cytoplasm.

In an in vivo approach rats were exposed to aqueous dispersions (administered by intratracheal instillation) or nanoparticle aerosols (inhalation) and alterations in the particle size distribution were studied using transmission electron microscopy (TEM) as well as the bronchoalveolar lavage (BAL) technique. Exemplarily, in BAL fluid after instillation, TiO2 P25 increased in agglomerate size whereas TiO2 T805 did not show a change as compared to the stock suspension.

As an additional endpoint, the chemical analysis of toxicokinetics was included to trace the fate of Eu2O3 particles following inhalative deposition in lungs. Only small Eu2O3 amountswere detected in remote organs.

Based on the results in various approaches, a tendency of nanoscaled particles to form larger size agglomerates following deposition and interaction with cells (in vitro) or the respiratory tract (in vivo) is predominant. The contrary trend, i.e. the increase of particle number due to a disintegration of agglomerates seems not to be of high relevance.

Key words:

Ultrafine primary particles, nanoparticles, dispersion, retention, lungs, particle characterisation, titanium dioxide, constantan, carbon black, amorphous silica, zinc oxide, europium oxide

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Dispersion und Retention von Stäuben mit ultra- feinen Primärpartikeln in der Lunge

Kurzreferat

In dieser Studie wurden die Dispersion und das Retentionsverhalten von Stäuben untersucht, die aus nanoskaligen Primärpartikeln bestehen. Toxikologische Studien haben gezeigt, dass die durch nanoskalige Partikel induzierten Effekte besser mit der Partikeloberfläche oder -anzahl korrelieren als mit der applizierten Partikelmasse.

Das toxikokinetische Verhalten nanoskaliger Partikel und die nach Deposition in der Lunge induzierten Effekte werden in erster Linie durch den Agglomerationsstatus bestimmt. Systemische Partikeleffekte, d. h. Effekte auf andere Organe oder Gewebe als den Respirationstrakt, sind nur für nanoskalige Partikel vorstellbar.

In dieser Studie wurden verschiedene Typen nanoskaliger Partikel, d. h. Titandioxid, Testruß, Konstantan und Zinkoxid unter gut definierten Bedingungen in physiologisch kompatiblen Medien dispergiert oder als Aerosole generiert. Bei wässrigen Nanopar- tikelsuspensionen wurden der hydrodynamische mittlere Durchmesser und das ζ-Potential bestimmt, bei Aerosolen die Partikelanzahl oder -massenkonzentration und der Partikelmassenmedian des aerodynamischen Durchmessers (MMAD). Für wässrige Formulierungen von Nanopartikeln wurde Phosphatpuffer, manchmal in Verbindung mit Hilfsstoffen wie Rinderserumalbumin (BSA) oder Tween^® 80 (nicht- ionischer Surfactant) eingesetzt.

In einem In-vitro-Ansatz wurden ausgewählte humane Bronchial- und Alveolarepithe- lialzellinien wie auch Fibroblasten (gewachsen auf Membranen) von der apikalen Sei- te her verschiedenen Partikeltypen exponiert. Nach 1 Stunde wurden die Partikel mit dem TEM besonders auf der Zelloberfläche detektiert, während sie nach 24 Stunden vor allem im Zytoplasma lokalisiert waren.

In einem In-vivo-Ansatz wurden Ratten gegenüber wässrigen Dispersionen (appliziert durch intratracheale Instillation) oder Nanopartikelaerosolen (Inhalation) exponiert und die Veränderungen in der Partikelgrößenverteilung mit dem TEM sowie mit der bronchoalveolären Lavage (BAL) untersucht. Beispielsweise zeigte TiO2 P25 in der BAL-Flüssigkeit nach instillativer Applikation einen Anstieg der Agglomeratgröße während TiO2 T805 diese Veränderung im Vergleich zur Stammsuspension nicht zeigte.

Als zusätzlicher Endpunkt erfolgte eine chemische Analyse zur Toxikokinetik, um das Verhalten von Eu2O3-Partikel nach Deposition in der Lunge zu verfolgen. Nur geringe Eu2O3-Mengen wurden in anderen Organen als der Lunge gefunden.

Auf der Basis der Ergebnisse verschiedener Versuchsansätze wurde gefunden, dass nanoskalige Partikel nach Deposition und Wechselwirkung mit Zellen (in vitro) oder dem Respirationstrakt (in vivo) vorherrschend eine Tendenz zur Bildung größerer Agglomerate zeigen. Die gegensätzliche Tendenz, d. h. ein Anstieg der Partikelan- zahl durch Agglomeratzerfall, scheint von geringerer Relevanz zu sein.

Schlagwörter:

Ultrafeine Primärpartikel, Nanopartikel, Dispersion, Retention, Lungen, Partikelcha- rakterisierung, Titandioxid, Konstantan, Carbon black, amorphes Siliziumdioxid, Zinkoxid, Europiumoxid

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1 Introduction

1.1 Toxicity of Fine Particles versus Ultrafine Particles

The toxic effects of fine (microscaled) and ultrafine (nanoscaled) particulate aerosols following inhalative uptake in the respiratory tract have been investigated intensely.

Animal experiments in rodents have demonstrated that exposure of lungs to respir- able particles can lead to the induction of inflammatory reactions, fibrosis and the development of lung tumours, in particular in the rat model. The same effects have been as well observed in comprehensive epidemiological studies in various industrial sectors (i.e. in cohorts exposed to aerosols in mining industries and in particle production processes or to engine exhausts). The findings in rodents after inhalation have been confirmed in parallel studies using the intratracheal instillation technique to administer particle suspensions. There were indications already in the nineties that the particle surface is one important factor in determining the carcinogenic potential of particles (OBERDÖRSTER et al., 2005; DUFFIN et al., 2007). There is agreement that nanoscaled particles or aggregates/agglomerates consisting of nanoscaled primary particles exhibit a higher toxic potential than microscaled particles of the same chemistry. Therefore, nanoscaled particles have not been classified according to the General Threshold Limit Value ("Allgemeiner Staubgrenzwert") in Germany. The biological activity of nanoparticles correlates better with the specific surface or number dose than with the mass dose.

1.2 Background

In nanoparticulate bulk materials the nanoparticles are usually present as clusters of aggregates and/or agglomerates (Fig. 1.1). While primary nanoparticles within aggregates are bound by strong chemical bonds (i.e., covalent or ionic bonds), binding between agglomerates is caused by van der Waals forces, which are less stronger. A well-characterised example is TiO2 P25 consisting of aggregates that are formed by sintering during the thermal production process. Therefore, the minimum size for TiO2

P25 agglomerates upon dispersion is limited to 90-100 nm. Smaller hydrodynamic diameters can merely be achieved albeit applying strong mechanical forces (MAY- NARD, 2002).

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Primary nanoparticle

Agglomerates of NP (held by weak van der Waals forces between particles)

Aggregates of NP (held by strong bonds between particles)

Agglomerated aggregates (held by strong bonds between aggregates and weak bonds between agglomerates)

Fig. 1.1 Aggregation and agglomeration of nanoparticles NP may be suspended

• in liquids

o in vitro for the investigation of physicochemical parameters, interaction with components of the liquid (e.g., salts, proteins, phospholipids) or biological materials (e.g. toxic effects on cells), or

o in vivo following the deposition of aerosols in the respiratory tract and, thereby, coming in contact with the liquid layer in the airways (especially, the surfactant layer in the alveoles of the lung);

• in air (aerosols)

o by dispersion of commercially available dry powder, the aggregation status being affected by the method and efficiency of dispersion, or o as freshly produced particles produced by dispersion of suitable materi-

als under controlled experimental conditions, thus enabling to modify the size and aggregation of the particles, or

o as mixed nanoparticle/buffer substance "particle". These mixed particles occur following nebulisation of a nanoscaled particle suspension after evaporation of the droplets. These mixed particles enhance the probability to obtain a real nanoscaled re-dispersion after deposition on the lung lining fluid.

In each case, the agglomeration or de-agglomeration of NP will be affected by the conditions of exposure and this, in turn, will affect the interactions with and the effects on the biological system.

Aim of the Project:

• study of agglomerates of nanoparticles following uptake in the respiratory tract with respect to disintegration into primary particles,

• identification of cell types responsible for uptake of these particles,

• systemic availability of nanoparticles from deposited agglomerates.

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Studies:

• in vitro identification of nanomaterials with suitable agglomerate size and

• inhalation and intratracheal exposure of animals to agglomerates of various defined sizes to study their further behaviour after prolonged time (> t½ clearance)

• focus on agglomerates, not aggregates.

1.3 Generation, Behaviour and Stability of Nanoparticles and Fate after Uptake in Lungs

Aerosols of nanoparticles

Aerosols really existing of airborne nanoparticles can be produced in very small mass but high number concentrations using a spark generator. For certain time periods (minutes) these nanoaerosols are stable, however, an ageing process is starting resulting in an increase of the agglomerate size. This experimental set-up of fresh particle generation together with ageing allows to expose the respiratory tract of animals to nanoscaled aerosols of a well defined size. The approach is often used in the ba- sic research field to deposit analytical (not toxicologically relevant) masses of nanoparticles in lungs and to analyse the toxicokinetic behaviour of those nanoparticles.

In contrary, aerosols generated by dispersion of nanoscaled bulk powders using pressurised air principally result in microscaled nanoparticle agglomerates the deposition of which is identical to that known for fine particles of the same size. The deposition of agglomerates is simply determined by the actual mass median aerodynamic diameters (MMAD) and the agglomerate density in the given experiment.

However, after deposition in the respiratory tract, those agglomerates may disinte- grate and release partially nanoparticles. If this happens this may have a high impact on the potential toxic effects in the respiratory tract and a systemic effect after translocation of nanoparticles.

Suspension of nanoparticles in aqueous systems

The dispersion status of nanoparticles in aqueous media principally depends on the surface properties of the given nanoparticle (e.g. hydrophilic particles are better dis- persable than hydrophobic ones). However, the dispersion of particles with hydrophobic surface can be highly improved by additives such as polyalcohols (e.g.

TWEEN 90^®). Sticky nanoparticles with hydrophilic surface can be dispersed more effectively by addition of proteins (e.g. bovine serum albumin - BSA). BSA is able to stabilise nanoparticle suspensions by a steric effect of the protein.

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2 Literature Search

As a basis for the project in a first step information from relevant primary literature on agglomeration/ deagglomeration of nanosized particles was obtained and evaluated.

A search in MEDLINE was performed with the following search terms and conditions:

762 hits were obtained, most of them after adding the terms "in vitro" and "in vivo".

Further selection of studies with probable or possible relevance for this project (based on title or abstract) gave 54 studies; some of them were already identified in previous searches (see Interim Report of December 2009 and application of project November 2008).

Finally, only studies with manufactured nanoparticles (NP in a strict sense of the definition of nanoparticles) were evaluated, not those with ultrafine particles formed as by-products in other (e.g. diesel particulate matter). Only studies with spheric NP, not with nanotubes (e.g. MCNT or SWCNT) or dendrimers etc. were included. The evaluation and overview focussed on studies that were primarily conducted to char- acterise the aggregation/agglomeration behaviour of NP, not on toxicity studies.

Only one in vivo study was identified which explicitly addressed the question − using two commercially available products of identical chemical composition − whether the size of the primary nanoparticles or the agglomerates seems more important in the inhalation toxicokinetics of nanoparticles (PAULUHN, 2009). The study is described in Appendix I (section 6.1.2).

2.1 Nanoparticles in Liquids

A number of in vitro studies with different nanoparticles have been published in which the effects of the following parameters on the dispersion, surface properties, and the agglomeration and de-agglomeration of nanoparticles were studied:

• ultrasonication,

• ionic strength and pH of aqueous solutions, physiological buffers, cell culture media,

• dispersion aids, both synthetic, e.g. Tween and DPPC, and from biological sources: proteins (especially albumin), serum, and lung surfactant.

The following chapter presents a brief summarizing overview of the methods applied and the results and conclusions that can be drawn from these studies. For details of each study and results for different nanoparticles materials, the reader is referred to Appendix I.

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2.1.1 Particle Characterisation

The aggregation and agglomeration of insoluble particles in biological fluids is an important issue in in vivo and in vitro toxicity studies. Such dispersions need to be characterized with respect to the size and size distribution of the particles, the stability of the dispersions and the effects of various methods and additives on these parameters.

An indicator for the tendency of particles in fluids to agglomerate is the zeta (ζ) potential: The zeta potential is the electric potential difference between the dispersion medium and the stationary fluid layer surrounding the dispersed particle (Fig. 1.1), including the conceptual "sliding surface". As this electric potential decreases towards zero, particles tend to agglomerate due to van der Waals forces overcoming electrostatic repulsion. On the other hand, higher zeta potentials will cause colloidal systems to remain stable and dispersed. However, the stability of dispersions in biological fluids is also determined by other, steric effects due to adsorption of proteins or other, surface-active substances. These effects have been investigated in several publica- tions.

Fig. 2.1 Double layer and surface charges surrounding a nanoparticle suspended in electrolyte containing medium¹

For the determination of the average size and size distribution of the dispersed particles and hence, by comparison with the size of individual nanoparticles, for an assessment of the agglomeration/aggregation of the dispersed particles, different methods are available. The size and aggregation of nanoparticles prior to dispersion can be determined by TEM or SEM. The most widely applied method for the size determination of dispersed nanoparticles is dynamic light scattering (DLS) in the corresponding medium. However, this method may have limitations, at least in protein- containing media, to detect small nanoparticles and may be biased towards large particles. A comparison of different methods showed that analytical ultracentrifuga- tion (AUC) may be the most reliable commonly available method to detect the pres-

1 www.symphotic.com/images/ZetaPotentialDiagram.gif

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ence of small-scale nanoparticles, although this method has limitations regarding the quantification of particles (MEIßNER et al., 2009; SCHULZE et al., 2009).

2.1.2 Ultrasonication

Ultrasonication of the test material has been a standard procedure to disperse poorly soluble or insoluble chemicals in biological fluids, and it is also commonly used in studies with dispersed nanomaterials (most of the studies of NP described in Appen- dix I included an initial sonication step in the preparation of the dispersions). The energy provided by ultrasound to form evacuated cavities can be capable to overcome the van der Waals forces between nanoparticles agglomerates, but is not strong enough to break up the stronger covalent or ionic chemical bonds of aggregates. Ul- trasonication has therefore been proposed and applied as a method to determine and distinguish between agglomeration and aggregation of dispersed nanoparticles (JIANG et al., 2009).

MAIER et al. (2006) observed that commercially available TiO2 nanoparticles (Aerox- ide^® P25, Degussa/Evonik) are highly aggregated and agglomerated in the original (aerosol) state. This microstructure was retained in aqueous dispersions without sonication, even in the presence of DPPC, and DLS measurements showed a monodisperse particle size distribution almost exclusively in the 1 – 10 µm range.

However, after bath ultrasonication of samples in PBS + DPPC an additional but small fraction with sizes around 100 nm became detectable. In accordance with these observations, JIANG et al. (2009) observed that the average size of the same TiO2 nanoparticles material could be reduced by ultrasonication. Nevertheless, the mean hydrodynamic diameter of these particles was still higher (155 nm) than the size of the individual nanoparticles, indicating that the material consisted of aggregates that could not be broken up by the treatment. In contrast, a laboratory prepared TiO2 of agglomerated but not aggregated primary particles with similar primary size could be dispersed by ultrasonication to nanoparticles of a mean hydrodynamic diameter of about 70 nm. Taking into account the electrical double layer around the primary particle, this size is consistent with the size of individual nanoparticles (JI- ANG et al., 2009). The results of other studies also confirm that the average size of TiO2 and other nanoparticles (zinc oxide, silver, silicon oxide) in dispersions and also the size distribution (as indicated by the PdI) may be reduced by ultrasonication of sufficient ultrasound energy. As may be expected, the exact reduction of the average size of nanoparticle agglomerates/aggregates in dispersions which may be achieved by ultrasonication depends on the chemical composition and on the method of preparation of the individual nanomaterial (BIHARI et al., 2008; TANTRA et al., 2010).

2.1.3 Effects of Ionic Strength, pH, Physiological Buffers, and Cell Culture Media

The effects of ionic strength, pH, and ion charge on electrostatic stabilisation on surface charge and agglomeration were investigated by JIANG et al. (2009). For this purpose, a 15 nm anatase TiO2 was freshly synthesized via a flame aerosol reactor from titanium isopropoxide. This material, dispersed in water or very diluted 1 µm NaCl solution, showed an average hydrodynamic diameter (by DLS) of 90 nm, which – taking into account the electrical double layer (see above) – is not much larger than the primary, non-aggregated, non-agglomerated nanoparticles. Dispersion in NaCl solutions of increasing ionic strength (1 – 100 mM, pH 4.6) increased the hydrody-

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namic diameter up to 50-fold (from ~ 90 to ~ 4800 nm). The dispersion became polydispers at 10 mM NaCl (with particle sizes mostly around 1000 nm); large, unstable and highly agglomerated dispersions were formed at NaCl concentrations ≥ 100 mM. The ζ potential of about +40 mV was not affected at low ionic strength but, consistent with the observations on the agglomeration, decreased at NaCl concentrations ≥ 10 mM. At constantly low ionic strength (1 mM), the ζ potential decreased with increasing pH. The hydrodynamic diameter reached its maximum at the isoelectric point of pH 6.0. Stable dispersions of small-sized particles (~ 90 nm) were obtained at pH< 4.2 or pH > 8.2.

The addition of 10 mM pyrophosphate (Na2P4O7) to TiO2 dispersions changed the ζ potential from ~ +40 mV to ~ -53 mV, because these ions are adsorbed on the surface of the particles. Consequently, this lead to an increased stability of the dispersion, so that no agglomeration occurred in solutions of 10 mM NaCl + 5 mM pyrophosphate. However, the stabilising effect of the polyvalent anion was limited to solutions of low ionic strength, and agglomeration could not be inhibited at 50 mM NaCl.

In accordance with these data, MEIßNER et al. (2009) observed that dispersions of TiO2 had a high positive ζ potential and were stable to agglomeration at acidic conditions and low ionic strength, but that the potential decreased in physiological NaCl, buffer solutions (PBS, HBSS) and cell culture medium (DMEM), and the nanoparticles rapidly agglomerated. A number of further studies with TiO2 from various sources and aggregate/agglomerate state, and also with other metal oxide and metal nanoparticles are in line with these observations. All these studies described that nanoparticle dispersions agglomerate in physiological salt solutions, e.g. TiO2 of various sources and characteristics (ALLOUINI et al., 2009; BIHARI et al., 2008;

MEIßNER et al., 2009; MURDOCK et al., 2008; SAGER et al., 2007; SCHULZE et al., 2008; TANTRA et al., 2010), ZnO (BIHARI et al., 2010; TANTRA et al., 2010), Al2O3 and Al (MURDOCK et al., 2008), some forms of SiO2 (BIHARI et al., 2008, MURDOCK et al., 2008), Ag (BIHARI et al., 2008; MURDOCK et al., 2008), Cu (MURDOCK et al., 2008), and carbon black (MURDOCK et al., 2008; TOTSUKA et al., 2009).

Therefore, the results of JIANG et al. (2009) and MEIßNER et al. (2009) are valid for nanoparticles in a general way and imply important consequences for dispersions of nanoparticles in physiological media. As such media must inevitably meet certain criteria with respect to pH, ion composition and ion concentrations, their composition will generally favour the agglomeration of nanoparticles. Consequently, it can be con- cluded that it will be very difficult or impossible to obtain stable dispersions of un- modified nanomaterials in physiological salt solutions including cell culture media. An inhibition or a limitation of the agglomeration may be achieved by surface coating of the nanomaterials, leading to electrostatic and steric stabilisation.

2.1.4 Effects of Dispersion Aids

LIMBACH et al. (2005) observed that different metal oxide nanoparticles showed different, substance specific ζ potentials in dispersions in pure water, but similar, slightly negative values in serum containing cell culture medium. Later studies showed that a similar change of the ζ potential can be observed in cell culture media regardless of the presence or absence of protein (MEIßNER et al., 2009; SCHULZE et al., 2008).

LIMBACH et al. (2005) postulated that, because the measured ζ potentials were too low to prevent agglomeration, a rapid agglomeration of nanoparticles would be favoured in such media. However, the mentioned studies (MEIßNER et al., 2009;

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SCHULZE et al., 2008) and a number of others revealed that dispersions of nanoparticles in protein-free culture mediums are unstable and larger agglomerates are rapidly formed, but that an agglomeration is in most cases inhibited or at least reduced in media containing proteins.

As the ζ potential of the nanoparticles in media with added proteins is rather small, the observed stabilisation of the dispersions cannot be explained by electrostatic effects. Instead, the stabilisation indicates a coating of the particles by proteins, leading to steric effects which stabilise the dispersion (JIANG et al., 2009; SCHULZE et al., 2009). Calculations for various metal oxide nanoparticles dispersions in serum containing cell culture media indicated that the resulting protein/metal oxide particles mainly consist of proteins and only smaller amounts of the metal oxide (1 – 43 %, depending on the metal oxide) (KATO et al., 2009, 2010).

In most studies, albumin has been used as a protein to stabilise NP dispersions, with no substantial differences between albumin from different species including humans (BIHARI et al., 2008). Also, results were generally comparable for albumin and for sera from different species (fetal calf serum, horse serum, mouse serum) (ALLOUINI et al., 2009; BIHARI et al., 2008; GOEBBERT et al., 2009; MEIßNER et al., 2009;

SAGER et al., 2007; TANTRA et al., 2009; VIPPOLA et al., 2009). Divergent results were obtained by MURDOCK et al. (2009) as the average particle diameter of various TiO2 and other nanoparticles was not reduced in medium with FCS compared to FCS-free media. The reasons for the discrepancy to other studies are not fully clear, but may be related to different sources and thus properties of the TiO2 and other materials. In any case, the results stress the need for rigid characterisation of particles in dispersion.

Fewer studies have investigated the effect of other dispersion aids on the agglomeration of dispersed nanoparticles. The dispersion of TiO2 and of carbon black in BAL fluid from rat lung (lavage wash with Ca²⁺- and Mg²⁺ free PBS) was described to consist of smaller agglomerates and show a more uniform dispersion pattern compared to PBS; however, the validity of these data is limited as only light microscopy observation was performed which cannot detect submicron particles (SAGER et al., 2007).

Using more precise and sensitive analytical determinations (analytical ultracentrifuga- tion), GOEBBERT et al. (2009) observed that the agglomeration state of nanoparticles (TiO2, AlOOH) in BAL fluid (porcine bronchoalveolar lavage fluid) was similar to that in diluted serum. Differences between this type of BAL fluid and diluted serum were observed for two cerium dioxide nanoparticle materials, where small-scale aggregates/agglomerates could only be detected in diluted serum (GOEBBERT et al., 2009).

Some data are available for DPPC as surrogate lung surfactant. TEM observations of technical TiO2 "as is" and after dispersion in DPPC revealed that the particle size decreased by a factor of three in dispersion. However, the particles were still about 5- fold larger (100 nm) than the primary particles (25 nm). These results suggest that the production process of ultrafine TiO2 nanoparticles led to the formation of primary stable aggregates of about 100 nm size and that larger agglomerates present in airborne suspensions may be broken up by DPPC to these aggregates but not to smaller ones or to individual nanoparticles (MAYNARD, 2002, 2008). This observation is in agreement with theoretical calculations and experimental data from MAIER et al. (2006) who found that the interaction energy between DPPC and the TiO2 surface is too low to overcome the intra-aggregate splitting energy between the nanoparticles.

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Very limited data were available regarding the effects of natural lung surfactant on the agglomeration status of nanoparticles dispersions. In one study, anatase-TiO2

dispersed in pure FCS with added natural lung surfactant (Curosurf) revealed an even distribution of agglomerated particles of a mean size similar to those observed in other media (VIPPOLA et al., 2009). However, the validity of these data is limited as only light microscopy observation was performed which cannot detect submicron particles Limited data are also available for Tween^® 80, a non-ionic synthetic emulsi- fier. E.g., TEM observations of a dispersion of carbon black in physiological saline with Tween^® 80 showed that carbon black was "relatively well dispersed, … agglomerates were occasionally present.", and the range of size distribution indicated that individual nanoparticles may have been present in the dispersion (TOTSUKA et al., 2009). In a study looking for optimised dispersion conditions for various nanoparticles, Tween^® 80 had similar effect as albumin with respect to size distribution and stabilisation of TiO2 dispersions. Also, the ζ potentials become rather similar in such dispersions with Tween^® 80 or albumin. Tween^® 80 was also as or slightly less effective in case of dispersions of silicon oxide and silver nanoparticles. However, substance specific differences have to be considered as Tween^® 80 was not as effective as albumin for dispersions of ZnO in PBS (BIHARI et al., 2008). In summary, these data indicate that the dispersion stabilising effect of Tween^® 80 seems largely comparable to that of proteins.

In conclusion, the following aspects are important to achieve a dispersion of nanoparticles in physiological solutions at a size of agglomerates/aggregates as small as possible (BIHARI et al., 2009): At the first step, the particles should be dispersed in water or – depending on the surface characteristics of the material – a very diluted acid or alkaline, so that the pH of the solution will favour a high positive or negative ζ potential which stabilises the dispersion against agglomeration. The dispersion should then be ultrasonicated using a sonication energy sufficient to break-up agglomerates, before albumin or serum is added as a stabiliser. The ratio of the concentration of protein to the concentration of nanoparticles must be high enough to ensure that the nanoparticles will be covered by the protein. As an alternative to albumin or serum, other stabilisers, especially Tween^® 80, may be used in experiments where the use of proteins is not appropriate, e.g., in studies with in vivo instillation of the dispersions into the airways. Finally, the dispersions may be diluted to the desired concentrations using the appropriate medium. The resulting dispersion should be characterised with respect to the average size and size distribution. This is usually done by DLS, the most widely applied method, and by TEM. However, it must be recognized that the results of DLS for dispersions in the presence of proteins may be biased. AUC has been suggested as a suitable, conventionally laboratory available method (SCHULZE et al., 2009). More work will be necessary to develop methods for precise and reproducible determination of the size and size distribution of dispersed nanoparticles.

2.2 Nanoparticle Suspension in Air 2.2.1 General Findings

During the production or airborne transportation in pipes, agglomeration is favoured when high particle concentrations and pressures are prevailing. Such agglomerates may be emitted in case of leakages and shear forces may cause break up of ag-

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glomerates. STAHLMECKE et al. (2009) observed that under experimental conditions simulating such leakage, nanoparticles agglomerates (< 100 nm) indeed partially fragmented with the formation of smaller particles (< 100 nm). The fraction of smaller particles increased with increasing overpressure. All materials contained a fraction of about 1 % of particles with a mobility diameter of < 100 nm. No significant fragmentation under overpressure conditions was observed for SrCO3, increases of approx. 3 – 6 % were observed for TiO2, TiZrAlO and a CeO2 material, all with primary particle sizes within 14 – 42 nm. In another CeO2 (70 nm) the fraction of particles < 100 nm increased at 12 %. The results of this study show that fragmentation to smaller particles may occur in case of leakages and that the amount of fragmented particles formed is somewhat dependent on the material.

2.2.2 In vivo Studies

In an inhalation study (28 days exposure, 3 months postexposure duration) with rats with two calcined aluminium oxyhydroxides the influence of primary particle size or the size of agglomerates on pulmonary effects (toxicity and fate) was investigated (PAULUHN, 2009) with identical particle mass concentrations in the exposure at- mosphere (Table 2.1, Appendix I).

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Table 2.1 Behaviour of agglomerates of nanoparticles in vivo γ-AlO(OH), Boehmite Particle

Pural 200 Disperal Dose levels mg/m³ 0.4, 3, 20 0.4, 3, 20 Particle characterization

Primary particle size nm 40 10

Particle size, powder d50 (measured)

µm 40 25

MMAD of aggregates µm 0.6 1.7

Surface Area m² 105 182

Kinetic parameters

Deposited alveolar fraction 0.103-0.105* 0.063-0.065*

t_1/2of elimination 56-144* 42-295*

Distribution PED 1, 28 mg/m³

- Lung % 83.2 80.3

- BAL Cells % 19.1 23.7

- Lymph Nodes % 1.1 0.1

PED 91, 28 mg/m3

- Lymph Nodes % 13.3 3.8

Toxicity

Effects only at 28 mg/m³

PMN + +

Inflammation, histopathology + +

*: Ranges for increasing dose levels; PED: Post Exposure Day.

Higher values are shaded in grey

Disperal consisting of the smaller primary nanoparticle (10 nm) formed larger agglomerates with a MMAD of 1.7 µm (Disperal 10/1.7), while Pural (40 nm) formed smaller agglomerates of 0.6 µm MMAD (Pural 40/0.6). Alveolar deposition was determined by the size of the agglomerates and was about twofold higher for the smaller agglomerates (Pural) despite identical exposure concentrations, indicating that the agglomerates remained stable during deposition.

For Disperal (10/1.7) at the lower dose level clearance in the postexposure period was higher than for Pural (40/0.6), indicating, that here the size of the primary nanoparticles plays a role. The author assumes that the smaller size and the larger surface area of the 10 nm primary NP may have facilitated the dissolution of the par-

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ticles at this low dose. Consistent with the higher lung burden also higher translocation of Al into the lung-associated lymph nodes (LALN) was observed for Pural- 40/0.6. There were no clear differences between the inflammatory potency and histo- pathological alterations of the two AlOOH particle types, although Disperal-10/1.7 led to a slightly more pronounced and sustained influx of PMN in the BAL over the whole experimental period.

Overall, this publication shows that although there are differences in the pulmonary distribution of the agglomerates, the overall toxicity is rather similar. However, one has to bear in mind that differences in sizes may be too low, for discriminating properties of the particles: the size of the particles differed by a factor of 4, and that of the agglomerates by a factor of only 2.5. In order to study effects related to particle sizes below 100 nm dry dispersion techniques of powders are not suitable. For selected materials such as carbon, pure metals, alloys and others the spark generation method is useful as this methods allows control over the particle size in the range between 20 and 200 nm.

2.3 References

Allouni ZE, Cimpan MR, Hol PJ, Skodvin T & Gjerdet NR (2009) Agglomeration and sedimentation of TiO2 nanoparticles in cell culture medium. Colloids Surf. B Biointer- faces 68 (1):83-87

Bihari P, Vippola M, Schultes, S., Praetner M, Khandoga AG, Reichel C. A., Coester C, Tuomi T, Rehberg M & Krombach F (2008) Optimized dispersion of nanoparticles for biological in vitro and in vivo studies. Part. Fibre Toxicol. 5(14):1-14

Goebbert C, Hardinghaus F, Kampmann KH, Kroell M, Lehr C-M, Schäfer U, Schultze-Isfort C, Schultze C, Voetz M & Wohlleben W (2009) Particle synthesis and characterisation. In: Kuhlbusch TAJ; Krug HF & Nau K, (eds.) NanoCare: Health related Aspects of Nanomaterials - Final Scientific Report, DECHEMA e.V., pp. 9-21 http://www.nanopartikel.info/files/content/dana/Dokumente/NanoCare/Publikationen/

NanoCare_Final_Report.pdf

Jiang J, Oberdörster G & Biswas P. (2008) Characterization of size, surface charge, and agglomeration state of nanoparticle dispersions for toxicological studies. J.

Nanopart. Res. 11(1):77-89

Kato H, Fujita K, Horie M, Suzuki M, Nakamura A, Endoh S, Yoshida Y, Iwahashi H, Takahashi K & Kinugasa S (2009) Dispersion characteristics of various metal oxide secondary nanoparticles in culture medium for in vitro toxicology assessment. Toxi- col. In Vitro

Kato H , Suzuki M, Fujita K, Horie M, Endoh S, Yoshida Y, Iwahashi H, Takahashi K, Nakamura A & Kinugasa S (2009) Reliable size determination of nanoparticles using dynamic light scattering method for in vitro toxicology assessment. Toxicol. In Vitro 23 (5):927-934

Limbach LK, Li Y, Grass RN, Brunner TJ, Hintermann MA, Muller M, Gunther D &

Stark WJ (2005) Oxide nanoparticle uptake in human lung fibroblasts: effects of particle size, agglomeration, and diffusion at low concentrations. Environ. Sci. Technol.

39(23):9370-9376

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Maier M, Hannebauer B, Holldorff H & Albers P (2006) Does lung surfactant promote disaggregation of nanostructured titanium dioxide? J Occup Environ med 48:1314- 1320

Maynard AD (2002) Experimental determination of ultrafine TiO2 deagglomeration in a surrogate pulmonary surfactant: preliminary results. Ann. Occup. Hyg. 46(1):197- 202

Meissner T , Potthoff A & Richter V (2009) Physico-chemical characterization in the light of toxicological effects. Inhal. Toxicol. 21(1):35-39

Murdock RC , Braydich-Stolle L, Schrand AM, Schlager JJ & Hussain SM (2008) Characterization of nanomaterial dispersion in solution prior to in vitro exposure using dynamic light scattering technique. Toxicol. Sci. 101(2):239-253

Pauluhn J (2009) Pulmonary Toxicity and Fate of Agglomerated 10 and 40 nm Alu- minum Oxyhydroxides following 4-Week Inhalation Exposure of Rats: Toxic Effects are Determined by Agglomerated, not Primary Particle Size . Toxicol. Sci.

109(1):152-167

Sager TM, Porter DW, Robinson VA, Lindsley WG, Schwegler-Berry DE & Cas- tranova V (2007) Improved method to disperse nanoparticles for in vitro and in vivo investigation of toxicity. Nanotoxicology 1(2):118-129

Stahlmecke B, Wagener S, Asbach C, Kaminski H, Fissan H & Kuhlbusch TAJ (2009) Investigation of airborne nanopowder agglomerate stability in an orifice under various differential pressure conditions. J Nanopart Res 11:1625-1635

Schulze C, Kroll A, Lehr C-M, Schäfer UF, Becker K, Schnekenburger J, Schulze Isfort C, Landsiedel R & Wohlleben W (2008) Not ready to use - overcoming pitfalls when dispersing nanoparticles in physiological media. Nanotoxicology 2:51-61

Tantra R, Tompkins J & Quincey P (2010) Characterisation of the de-agglomeration effects of bovine serum albumin on nanoparticles in aqueous suspension. Colloids Surf. B Biointerfaces 75(1):275-281

Totsuka Y , Higuchi T, Imai T, Nishikawa A, Nohmi T, Kato T, Masuda S, Kinae N, Hiyoshi K, Ogo S, Kawanishi M, Yagi T, Ichinose T, Fukumori N, Watanabe M, Sugi- mura T & Wakabayashi K (2009) Gentoxicity of nano/microparticles in in vitro micro- nuclei, in vivo comet and mutation assay systems. Part. Fibre Toxicol. 6:23, 11p.

Vippola M, Falck GCM, Lindberg HK, Suhonen S, Vanhala E, Norppa H, Savolainen K, Tossavainen A & Tuomi T (2009) Preparation of nanoparticle dispersions for in- vitro toxicity testing. Hum. Exp. Toxicol. 28(6-7):377-385

Wagner AJ , Bleckmann CA, Murdock RC, Schrand AM, Schlager JJ & Hussain SM (2007) Cellular interaction of different forms of aluminum nanoparticles in rat alveolar macrophages. J. Phys. Chem. B 111(25):7353-7359

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3 Materials and Methods

3.1 Selection of Nanoscaled Particles

3.1.1 Commercially Available Nanoscaled Particle Pairs

Well-characterised and commercially available pairs of nanoscaled dusts were obtained from the sources given in Table 3.1: Titanium dioxide P25 (hydrophilic) and T 805 (hydrophobic); amorphous silica Aerosil 150 (hydrophilic) and R 104 (hydrophobic); technical soot Printex 90 (BET specific surface: 300 m²/g) and XE 2 (BET specific surface: 910 m²/g); zinc oxide (< 100 nm, not doped) and zinc oxide (< 50 nm, 6 % Al-doped); see also Appendix II.

Table 3.1 Selection of nanodust pairs

Substance Particle type Average primary particle size (nm)

Specific surface (BET) (m²/g)

Tapped density (g/cm³)

pH

AEROXIDE^® TiO2 P 25

Fluffy white powder

Hydrophilic fumed metal oxide CAS # 13463-67-7

EINECS # 236-675-5 Lot # 4168112198 * Evonik-Degussa

21 50 ± 15 0.13 3.5-4.5 Titanium

dioxide

AEROXIDE^® TiO2 T 805

Fluffy white powder Hydrophobic metal oxide

CAS # 100 209-12-9 ex. 13463-67-7 EINECS # 309-319-2 ex. 236-675-5 Lot # 3159021267

Evonik-Degussa

21 45 ± 10 0.2 3.0-4.0

AEROSIL^® 150

Fluffy white powder Hydrophilic fumed silica

CAS # 112945-52-5 ex. 7631-86-9 EINECS # 231-545-4

Lot # 3159020511 Evonik-Degussa

14 150 ± 15 0.05 3.7-4.7 Amorphous

silica

AEROSIL^® R 104

Fluffy white powder Hydrophobic fumed silica CAS # 68583-49-3 EINECS # 271-514-2 Lot # 3159031722 Evonik-Degussa

12 150 ± 25 0.05

(tamped)

> 4.0

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PRINTEX^® 90

Fluffy black powder High Colour Furnace Black CAS # 1333-86-4

Lot # 8313101 Evonik-Degussa

14 approx.

300

- - Technical

soot

PRINTEX^® XE 2

Fluffy black powder Extra Conductive Black CAS # 1333-86-4 Lot # 080725 Evonik-Degussa

30 approx.

910

- -

* This TiO₂sample is the official lot used in the TiO₂ OECD Sponsorship Programme

Table 3.1 Selection of nanodust pairs - cont'd Zinc oxide, nanopowder,

<100 nm

White powder Not doped CAS # 1314-13-2 EINECS # 215-222-5 Lot # 46097LJ Aldrich

- - - -

Zinc oxide

Zinc oxide/6 % Al doped, nanopowder, <50 nm (BET)

White powder Doped

CAS # 1314-13-2 EINECS # 215-222-5 Lot # MKBB0132 Aldrich

- - - -

Some of these powders have been frequently used in toxicological studies (e.g. TiO2

P25, Aerosil 150, Printex 90). Their agglomeration behaviour was compared to coun- terparts with contrary surface properties. This juxtaposition gave insight into the influence of surface modification on the particle size distribution in media and physiological ambience (e.g. lungs).

3.1.2 Generation of Nanoscaled Constantan Particles

Constantan is a copper-nickel alloy usually consisting of 55 % copper, 44 % nickel and 1 % mangane. Ultrafine constantan particles are generated in an argon atmos- phere using a modified electric spark generator (Typ GFG 1000, Palas, Germany) with two constantan electrodes. The reaction chamber of the particles generator is produced of inert ceramic to generate particles without contamination of organic components. The particle size distributions were in the range from 25-150 nm depending on the ageing for the freshly produced aerosol. Particle growth was achieved by agglomeration in a coagulation chamber. Thus, it was possible to expose animals in an acute inhalation tests to nanoscaled or non-nanoscaled aerosols of constantan.

In addition, absolute amounts of up to 600 mg of the freshly generated aerosol have been collected on filters in manifold days of processing and sampling (approx. 10-20 mg achievable per day (Table 3.2). This material was used also for i.) liquid agglomeration status work in vitro in physiological media and ii.) for the acute inhalation of constantan where the aerosol was generated by dispersing a constantan suspension in aqueous phosphate/bovine serum albumin solution.

Details of the constantan nanoparticle generation process are presented in Appendix III.

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Table 3.2 Constantan nanodust generated by spark erosion of a constantan electrode

Substance Particle type Average primary particle size (nm)

Specific surface (BET) (m²/g) Constantan Constantan (copper-nickel alloy

consisting of 55 % copper,44 % nickel and 1 % mangane; main feature: resistance which is constant over a wide range of temperatures)

Nanopowder sample prepared by electrode erosion at Fraunhofer ITEM

5-7 111¹

1 Calculated based on a material density fo 9 g/cm³ and a primary particle diameter of 6 nm

Note: During the BAuA-ITEM meeting on April 2, 2009 silver or copper were also discussed as candidates for the sparkling generation process; however, technical trials revealed that both metals where not optimal.

3.1.3 Commercially Available Nanoscaled Europium Oxide (Eu2O3)

Europium oxide is a metal oxide within the group of rare earths and was selected for an acute inhalation test because i.) nanoscaled fractions of Eu2O3 are commercially available, e.g. at americanelements.com, USA); ii.) its water solubility is negligible;

iii.) the background of Eu2O3 in biological matrices is very low thus allowing very precise determinations of potential translocation of deposited materials from lungs.

In addition to the toxicokinetic analysis TEM analysis was also included to verify whether race amounts of europium oxide found in remote localisations were based on dissolved or still particulate material.

An Eu2O3 specified as "99.9 % (REO) EuropiumOxide Nanopowder" sample (product code: EU-OX-03R-NP; lot #: 1271513379-620) was purchased from American Ele- ments Co., Los Angeles, CA, USA. This is an uncoated Eu2O3 with hydrophilic surface properties.

Table 3.3 Physical properties of Eu2O3

Formula CAS No.

Appearance Molecular Weight Density

Melting Point Boiling Point Solubility Stability

Eu₂O₃ 1308-96-9 White 351.92 7400 kg/m³ 2350 °C

Insoluble in water, moderately soluble in strong mineral acids

Slightly hygroscopic

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3.1.4 Determination of Particle Characteristics in Liquids with the ZetaSizer^®

Hydrodynamic Diameter (Z-average)

The particle size distribution was measured using a ZetaSizer^® (Malvern Instruments Ltd, UK). This device widely used in nanoparticle characterisation is based on dynamic light scattering (DLS). The time-dependent fluctuations of laser light intensity scattered by the particles (particles in Brownian movement) is measured and evaluated by an integrated software. The output using the cumulant method is an intensity- weighted harmonic mean of the particle size diameter which is derived from the slope of the autocorrelation function (Z-average). The Z-average value measured physically describes a mean value of the hydrodynamic particle size distributions. The dimen- sionless polydispersity index (PDI) descibes the width of the particle size distribution (0 = monodisperse; 1 = polydisperse). Further results derived by calculation are vol- ume (mass)- or number-weighted particle size distributions. For the calculation the refractive indes of the particle substance is required (e.g. for TiO2 = 2.55; not for all substances data are available from the literature, for those the values have to be taken from adequate analogues).

ζ potential

The ζ potential was determined by measuring the electrophoretic mobility of particles in suspension. The ZetaSizer^®performs a calculation of the ζ potential (Smolu- chowski equation). For this measurement specific capillary cells are used.

3.1.5 Determination of Particle Characteristics in Aerosols Spark generator (constantan)

The generator was operated with an argon flow rate of 3.5 l/min; in case of additional ageing was passed through a 10 l ageing cylinder. It was then further diluted by 20 l/min compressed air and entered the inhalation chamber.

The average size distribution was characterized by a mean mobility diameter of 124 nm and a geometric standard deviation of 1.9. For details of conditions in the acute inhalations refer to section 4.2.2.

Nebulisation of nanoparticle suspensions (constantan and europium oxide)

The particulate sample aerosol was generated by dispersing the aqueous suspensions. After evaporation of water from the droplet aerosol the aerosol concentration were determined by taking filter sampling as well as on-line aerosol photometer re- cording.

The mass median aerodynamic diameter (MMAD) was determined using a Marple impactor.

3.2 Preparation of Particle Suspensions 3.2.1 Inititial Considerations

The suspension of nanoscaled particles to be used for in vitro cell incubations and in vivo intratracheal instillation experiments should use physiologically compatible buffer media. For the first nanoparticle pair, i.e. TiO2 P25 and TiO2 T805, a phosphate

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buffer was chosen that had been successfully used in a nose-only experiment in rats to generate a partially nanoscaled TiO2 P25 aerosol (Fraunhofer ITEM study no. 02 N 08 522; Creutzenberg et al., 2009; see Appendix V). In case of TiO2 T805 (hydrophobic surface properties because of surface-treatment with organics) Tween^® 80 was used as an additional detergent auxiliary.

Besides phosphate buffer other media were tested, e.g. the commercially available surfactant Curosurf^® as an auxiliary for dispersion purposes.

Curosurf^®: Phospholipid fraction from swine lung; 120 mg corresponding to 111 mg total phospholipid; 1.5 ml Suspension; sodium chloride, sodium hydrogen carbonate (Chiesi GmbH, Hamburg; lot # 096941; expiry date: 10/2010).

Porter et al. (2008) described a dispersion medium (DM), i.e. a Ca^{2 +} and Mg²⁺-free phosphate buffered saline (PBS), pH 7.4, supplemented with 5.5 mM D-glucose, 0.6 mg/ml species-specific serum albumin, and 0.01 mg/ml 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC). This medium can be used as a 'lung fluid mimic.'

The SOPs edited during the NanoCare Project have also been considered. However, they have not been used for this project as they focus on workplace situations and aim at investigating the given powders (agglomerates) more than really existing nanoparticles.

3.2.2 Preparation of TiO2 P25 Suspensions Sequence of treatment (general procedure)

- Suspension of TiO2 P25 (0.1 w-%) in a 0.15 w-% Na2HPO4 buffer - 30 min treatment with Ultra-Turrax (high shear forces)

- 30 min ultrasonic treatment (high dis-aggregation forces)

Table 3.4 Measurements of hydrodynamic diameter and ζ-potential with TiO2 P25 TiO2 P25

Sample treatment

Time log (h)

Hydrodynamic diameter

(nm)

ζ-Potential (mV)

after 30 min Ultra-Turrax treatment 0.5 205.6 -52.6 after additional 30 min ultrasonic treatment 1 181.7 -50.3 after additional 4 hrs without treatment 5 181.0 -49.9 overnight without treatment 20 152.0 -53.0 after additional 4 hrs without treatment 24 177.9 n.m.

addition of BAL fluid (1:1 v/v) 24 + 5 min precipitate! n.m.

- BAL fluid: - obtained by 2x4 ml lung lavage with saline from an untreated rat; - 10 min centrifugation at 3000 rpm of the BAL fluid to remove the cells

- Measurements done with ZetaSizer^®, Malvern, England

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3.2.3 Preparation of TiO2 T805 Suspensions Sequence of treatment (general procedure)

- Suspension of TiO2 T805 (0.1 w-%) in 0.15 w-% Na2HPO4 buffer/0.09 w-%

Tween 80

- 30 min treatment with Ultra-Turrax (high shear forces) - 30 min ultrasonic treatment (high dis-aggregation forces)

Table 3.5 Measurements of hydrodynamic diameter and ζ-potential with TiO2 T805 TiO2 T805

Sample treatment

Time log (h)

Hydrodynamic diameter

(nm)

ζ-Potential (mV)

after 30 min Ultra-Turrax treatment 0.5 183.0 -16.7 after additional 30 min ultrasonic treatment 1 175.0 -17.2 after additional 4 hrs without treatment 5 177.9 -16.9 after addition of Curosurf (0.05 w-%) 5 + 5 min 177.2 -18.8 after additional 19 hrs without treatment 24 177.9 n.m.

addition of BAL fluid (1:1 v/v) 24 + 5 min 191.6 n.m.

- BAL fluid: - obtained by 2x4 ml lung lavage with saline at an untreated rat; - 10 min centrifugation at 3000 rpm of the BAL fluid to remove the cells

- Measurements done with ZetaSizer^®, Malvern, England

3.2.4 Preparation of Carbon Black Particle Suspensions

Carbon black (Printex^®90) particles were dispersed in the 'lung fluid mimic' (0.1 w-%

carbon black) described by Porter et al. (2008). The following conditions were used to generate a nanoscaled or microscaled carbon black suspension:

• Nanoscaled: 50 min vigorous stirring with the Ultra Turrax^® (IKA Co.) followed by approx. 22 min ultrasonic treatment

• Microscaled: 8 min vigorous stirring with the Ultra Turrax^® (IKA Co.) followed by approx. 13 min ultrasonic treatment

3.2.5 Preparation of Zinc Oxide Particle Suspensions

Zinc oxide suspensions were prepared as given in the following table.

Table 3.6 Measurements of hydrodynamic diameter and ζ-potential with ZnO powders

Substance / particle type

Concen- tration

(%)

Medium Z-Average (nm)

ζ potential (mV) ZnO < 100 nm 0.1 0.15 % Na2HPO4 buffer 265 -62

after 20 h 222 -65

ZnO < 50 nm 220 -53

after 23 h 0.1

0.15 % Na₂HPO₄ buffer

0.05 % Tween 80 218 -55

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3.2.6 Preparation of Constantan Particle Suspensions

Constantan particles were dispersed in 0.15 % phosphate buffer supplemented with bovine serum albumin (BSA).

For the intratracheal instillation experiments a concentration of 0.2 w-% constantan/0.15 w-% Na2HPO4/0.15 w-% bovine serum albumin (BSA) was used. The suspension was treated vigorously for 40 min using the Ultra Turrax^® (IKA Co.) followed by short ultrasonic treatment and manual shaking.

For the inhalation experiment a composition of 0.15 w-% constantan/0.15 w-%

Na2HPO4/ 0.15 w-% bovine serum albumin (BSA) was chosen for the stock suspension.

3.2.7 Preliminary Conclusion

Various approaches applied in the project demonstrated that it is feasible to prepare stable, at least partially nanoscaled suspensions of the selected nanoscaled powders in physiological media. The particle suspensions generated with a strong mechanical treatment and using various methods in combination, showed an evident moiety of particles with a diameter below the 100 nm threshold. This is evident after evaluation of the Z-average particle diameter data and, in particular, when calculating the number-weighted particle diameters.

Trials to prepare stable particle suspensions with a mean size in the range of 300- 1000 nm failed. It is difficult to establish mean size values in the microscaled particle range stable for substantial time periods.

3.3 In vitro Approaches

To gain deeper insights into the uptake and fate of the test particles, an exemplary in vitro approach with selected human cell lines from the respiratory tract was per- formed using an air-liquid interface culture.

3.3.1 Selection of Cell Lines

The selection of the cell models based on their acceptance in current research and aimed to represent each region of the respiratory tract. Therefore, the following three human epithelial cell lines from bronchial and alveolar localisation as well as one human lung fibroblast cell system were used:

Calu-3

This cell line is a human sub-bronchial gland cell line and features epithelial morphol- ogy as well as adherent growth (ATCC; 2010). Calu-3 cells form polarized confluent monolayers with tight junctions (Shen et al., 1994), and resemble ultrastructure, levels of m-RNA and protein content characteristics of the native epithelium (Finkbeiner et al., 1993). They were reported to contain secretory granules and express mucus genes (Gruenert et al., 1995; Shen et al., 1994; Berger et al., 1999). Due to there origin from sub-bronchial glands these cells produce mucus under air-interface conditions (O´Shaughnessy and Prosser 1996). Furthermore, cells exhibit several ion transport characteristics (Mathias et al., 1996; Shen et al., 1994) and express CYP- enzymes (Foster et al., 2000). The Calu-3 cell line has shown utility as an in-vitro

Dispersion and Retention of Dusts Consisting of Ultraﬁne Primary Particles in Lungs

Consisting of Ultraﬁne Primary Particles in Lungs

Research Project F 2133

Dispersion and Retention of Dusts Consisting of Ultrafine Primary Particles in Lungs

Contents

Dispersion and Retention of Dusts Consisting of Ultrafine Primary Particles in Lungs

Dispersion und Retention von Stäuben mit ultra- feinen Primärpartikeln in der Lunge

1 Introduction

2 Literature Search

3 Materials and Methods